Characterization of human adenovirus 35 and derivation of complex vectors
© McVey et al; licensee BioMed Central Ltd. 2010
Received: 9 July 2010
Accepted: 19 October 2010
Published: 19 October 2010
Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery.
Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.
The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.
Recombinant adenovirus (rAd)-based gene transfer vectors are currently under investigation in a variety of gene therapy and vaccine clinical trials. There are more than 370 such clinical trials that are ongoing for broad applications, including infectious diseases and cancer therapy http://www.wiley.com//legacy/wileychi/genmed/clinical/. Many of these trials are of recombinant adenovirus (rAd) vectors based on the human serotype 5 (Ad5) yet there are advantages to rAd vectors derived from other serotypes. The human adenoviruses have previously been shown to have different prevalence in populations around the world . Exposure of human populations to adenovirus serotype 35 (Ad35) has been shown to be relatively rare based on the prevalence of Ad35 neutralizing antibodies in sera [1–5]. Because neutralizing antibody could interfere with the efficacy of viral gene transfer vectors, the low seroprevalence of Ad35 makes it an attractive candidate for derivation of viral vectors [1, 4, 6, 7].
The Ad35 genome has an overall organization similar to all adenoviruses [1, 6, 8], which facilitated the derivation of E1-deleted rAd35 vectors. However, the reported Ad35 genome annotations were based on sequence homology analyses without experimental evidence and some of the initial rAd35 vectors were found to have genetic instability due to the inadvertent deletion of the promoter for the structural protein IX (pIX) . Thus, homology analysis was limited in predicting functional regions of the Ad35 genome. To develop a complex rAd35 vector with up to three large deletions we attempted to characterize the Ad35 life cycle relevant to rAd35 viral vector productivity, stability, and capacity for foreign DNA. Essential sequences were identified in E1 and E4, the sequences were deleted, and the effects of the deletions on viral gene transcription were determined. The non-essential E3 region was also deleted from rAd35 vectors and a sequence was found that unexpectedly affected late fiber gene transcription, with subsequent effects on viral fitness. The packaging capacity of rAd35 was dependent on pIX and viral capsids with pIX packaged viral genomes up to 104% of the wild type genome size, whereas pIX-deficient capsids had a packaging limit of less than 100% wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages over previous rAd35 vectors of a second block to viral replication (i.e., E1 and E4 deletions) and an expanded transgene packaging capacity totaling 7.6 Kb, comparable to rAd5 vectors.
Ad35 capsid components and identification of the early/late switch
Identification of Ad35 viral particle proteins.
Protein mass spectroscopy molecular weight (MW) determination*
Whole protein MW comparison
Tryptic peptide Identification
SDS-PAGE molecular weight**
Experimental, theoretical (Da)
No. of peptides
Size and abundance
II, n-term. fragment
8.50 × 10-13
Tryptic peptide fingerprint
II, c-term. fragment
Abundance in RP--HPLC
III (Penton Base)
6.30 × 10-6
SDS-PAGE of RP-HPLC fraction
2.10 × 10-9
SDS-PAGE of RP-HPLC fraction
Size shift of modified protein
2.10 × 10-6
Inference from MW
SDS-PAGE of RP-HPLC fraction
Inference from MW, silver and Deep Purple staining intensity
1.50 × 10-9
Inference from MW
Inference from MW and IX deficient virus
Characterization of the Ad35 E1B/pIX region
An objective for optimal rAd vector design was to maximize the space available in the genome for insertion of transgene expression cassettes. All coordinates of viral locations are based on the Ad35 Holden sequence (GenBank accession number AY128640) To design the largest deletion of the E1 region we determined if E1B and pIX transcripts were generated from overlapping genome sequences that would be perturbed by deletion of E1B. First, DNA sequencing of a λ cDNA library from Ad35-infected cells identified the polyadenylation site and the junctions for two introns. The cDNA clones that included E1B and pIX sequences showed polyadenylation occurring at the position equivalent to nucleotide 3952. Thus, the Ad35 E1B and pIX transcripts utilized the polyadenylation hexanucleotide signal at nucleotides 3925-3930 and 3929-3934 inclusive. The left-most splice junction identified in the cDNA library joined base pairs 2180 and 3231, placing it in the open reading frame previously annotated to encode the E1b 494R protein which is analogous to Ad5 E1B 55k . A second splice junction joined base pairs 3402 and 3480, placing it in the intergenic region between E1B and pIX coding sequences with the 3' splice junction (bp 3480) five nucleotides upstream of the pIX initiation codon. The proximity of the 3' splice junction implied that the promoter for pIX was located farther upstream than would have been predicted by homology to the well-characterized species C adenoviruses .
Ad35 viral vector packaging capacities.
E1 deletion, transgene
Other genome modifications
Genome size (percent of wt)
Stability at Passage 3
d2, no transgene
d2, green fluorescent protein (GFP)
Δ447 - 3326, GFP
d2, secretory alkaline phosphatase
d2, bacterial beta-glucuronidase
d2, HIV Env gp140ΔCFIΔV1V2
d2, HIV Env gp140ΔCFI
d5^, HIV Env gp140ΔCFIΔV1V2
d8, HIV Env gp140ΔCFI
d8, HIV Env gp140ΔCFI
The relationship of pIX expression to capsid integrity provided criteria for the selection of an optimal E1 deletion: maximum deletion of E1 sequences and retention of native control of pIX expression. The E1(d8) deletion was selected as the base for further rAd35 vector development. Recombinant Ad35E1(d8) vectors contained the largest E1 deletion tested that maintained wild type level of pIX transcription (transcript 'c'). In addition, the yield of rAd35E1(d8) viral progeny was consistently high, approximately 100,000 particles per cell when propagated on the 293-ORF6 cell line (data not shown).
The Ad35 E4 Open Reading Frame 6 sequence was necessary and sufficient for E4 complementation
Transcription analysis of the Ad35 E4 region with deletions
Ad35 E3 region deletions and effects on fiber gene regulation
Once the fiber transcript was identified, the effects of E3 region deletions on fiber transcription were determined. A panel of E3 deletions was generated in the E1-deleted backbone rAd35(d8) (Figure 7B), and fiber transcripts were analyzed in cells infected with the E1- and E3-deleted rAd35 vectors. The E3(X) deletion appeared to increase the level of both the 4.0 kb and 1.5 kb fiber transcripts and a new RNA of approximately 6.0 kb was detected (Figure 7A). Phosphoimager quantification of the 1.5 kb and 4.0 kb bands showed a 2-3 fold increase relative to wild type Ad35 and rAd35(d8) (data not shown), while the level of the pIX transcript did not change. In contrast, E3 deletions SB, HB and HE did not affect the quantity or size of the fiber transcripts (Figure 7A). Therefore, the sequences between 27,607 and 30,409 were not required for proper fiber transcription. However, sequences between 27,240 and 27,608 effected fiber transcription. A polyadenylation signal for L4 is present in this region  and the 6.0 kb transcript found with the E3(X) deletion may represent L4 transcript(s) using the fiber polyadenylation signal, even though the E3 polyadenylation signal was intact in E3(X). The change in relative amount of fiber mRNA could be reflective of the differential regulation of polyadenylation signal usage by the late transcription unit.
Despite the altered late gene expression, the rAd35(d8) vector with the E3(X) deletion was efficiently rescued and grew to high titers. However, the productivity of the rAd35(d8) with the E3(X) deletion was 2- to 3-fold lower than the E1-deleted rAd35(d8) (data not shown). The effects of the E3 deletions were further evaluated. The relative fitness of E1-deleted vectors with and without E3 deletions was determined in virus competition experiments by co-infecting 293-ORF6 cells with equal amounts of purified virus particles from two viruses and serial passaging the resultant cell-virus lysates three times. The presence of each viral genome was determined by restriction digest of total DNA harvested from each lysate. The E3(X) virus could not be detected in the lysate from the first infection (Figure 7C), whereas the E3(HE)-deleted vector genome persisted at its relative input level through the rounds of infections (Figure 7D). Therefore, the E3(X) deletion conferred a dramatic reduction in fitness relative to E1-deleted rAd35. In contrast, the double-deletion rAd35 vector E1(d8) with the largest E3 deletion, HE, showed no reduction in relative fitness and its viral productivity was comparable to rAd35 with only an E1(d8) deletion, 86,000 and 98,000 pu/cell respectively. Thus, the E3(HE) deletion maintained proper fiber transcription, viral fitness, and good virus productivity. The E3(HE) deletion provides 2801 bp of space for foreign DNA.
Generation of triple-deletion rAd35 vectors
Viral progeny yields of Ad35 vectors on 293-ORF6 cells
To further expand the utility of rAd35 vectors a triple-deleted vector was designed. The replication-deficient vector incorporated the deletions that had the least impact on virus function while maximizing the amount of space available for heterologous sequences. The combination of E1(d8), E3(HE), and E4(AN) provided these characteristics. In addition, rAd35 vectors with this combination of deletions yielded 36,000 pu/cell, which is comparable to the E1-deleted rAd35 (Table 3). The vector has the capacity to accommodate 6,347 bps of heterologous sequence and still remain at only 100% of wild type genome size. The vector genome was stable; no rearrangements were detected by the PCR assay in the E1, E3 and E4 regions after 10 serial passages of cell-virus lysate. In addition, protein composition of the capsid was found to be indistinguishable from wild type Ad35 in SDS-PAGE analysis (data not shown). This ease of construction, genetic stability, high productivity, reduced potential to generate a replication competent adenovirus (RCA) by homologous recombination with the production cell line, and expanded heterologous packaging capacity makes the triple-deleted vector configuration particularly suitable for use with large expression cassettes and commercial manufacturing purposes. To our knowledge this is the first E1-, E3-, E4-deleted rAd35 vector produced.
A combination of biochemical and biological approaches was used to derive multiple rAd35 vectors with the best replication capacity and ability to accept large insertions of DNA. Analysis of the regions of the genome targeted for deletion provided information for optimal vector design for rAd35 vectors with one, two, or three deletions. The construction of rAd35 viruses with E1 and/or E4 deletions and the analyses of the E1 and E4 regions were dependent on the 293-ORF6 cell line which provided E1 and E4 complementing activity. The 293-ORF6 cell line, which expresses Ad5 E1 and Ad5 E4 ORF6 gene products, has been shown to complement replication-deficient vectors derived from many serotypes [15–17].
Transcript and cDNA clone analysis provided for the identification of splice junctions and polyA sites for E1B, pIX, and fiber transcripts, as well as the major E4 transcripts. Previously, the pIX transcript was identified to have an intron . Here we identified the splice junctions and show the intron is common to E1B transcripts. This common intron had not been previously reported and may be a feature of adenoviruses that do not have a discrete promoter for pIX positioned between the E1B ORF and the pIX ORF as found in Ad5 . Transcript analysis of fiber gene expression revealed that deletion of non-essential E3 region sequence can effect fiber late gene expression. Deletion of the L4 polyA signal sequence  in the E3 region created a large new transcript that was generated from the fiber polyA signal, thus the E3 polyA signal was skipped, at least during the late phase of infection, and fiber transcription was increased, relative to wild type Ad35 levels. This demonstrates that disruption of gene expression regulation via deletion of non-essential sequences can result in viable but under-performing rAd vectors. Previously described E3-deleted Ad35 vectors removed the putative L4 polyA signal, however, no assessment of viral productivity or stability was provided .
The packaging capacity of adenovirus type 5 has been reported as 105% of the wild type Ad5 genome size . The resultant preparations of Ad5 with genomes of 105% were mixed populations of full length and genomes with spontaneous deletions, thus the large genome could be packaged into the Ad5 virion but there was a strong selection for shorter genomes. In contrast, the Ad35 packaging capacity reported here was defined by maintenance of genetic stability. Recombinant Ad35 vectors with wild type levels of pIX transcription could accommodate genomes up to 103.6% of wild type genome size without signs of deletions or other genetic instability.
A deletion of the E4 region provides advantages for rAd vectors. Ad35 E4 sequences were essential for replication and their removal provided a second block to replication beyond that obtained by deleting E1. Removal of the E4 region further diminishes the likelihood that the vaccine vector could revert to replication competency in the human host while simultaneously avoiding transformation potential . E1- E3-, E4-deleted rAd5 vectors display marked decreases in expression of other adenovirus and cellular genes, relative to first-generation vectors possessing only the E1 or E1/E3-deletion(s) [19, 20]. This property of the vector results in reduced toxicity to transduced cells both in vitro and in vivo  and may reduce the induction of adenovirus-specific T cell activation following immunization . The E1-E3-E4- rAd35 vector would be predicted to also have similar properties. Lastly, the E4 deletion provides an additional 1075 bp of space in the vector genome for insertion of foreign DNA. Thus, the triple-deletion backbone can stably accommodate 7,600 bp of foreign DNA, based on a genome size of 103.6%.
Ad35 was identified as an attractive candidate for gene transfer vectors because of hematopoietic stem cell tropism  and neutralizing antibodies to Ad35 are relatively uncommon in human populations. The frequency of individuals with pre-existing immunity to Ad35 was found to range from <5% in the USA and Europe to up to 20% in Africa [1, 3, 4]. Ad35 vectors have been extensively tested in animal models of vaccination, as well as in ongoing Phase I trials for HIV (ClinicalTrials.gov: NCT00479999, NCT00472719, NCT00801697), malaria (ClinicalTrials.gov: NCT01018459, NCT00371189), and tuberculosis (ClinicalTrials.gov: NCT01017536, NCT01198366). However, results from the preclinical studies of rAd35 vaccine vectors indicated that transgene-specific immune responses were markedly lower compared to Ad5 vectors [22–24]. The lower immunogenicity of rAd35 vectors may have benefits for gene therapy applications because administration of rAd35 vectors to the eye gave prolonged transgene expression relative to Ad5 vectors . In summary, the data reported here expanded the knowledge of adenovirus 35 and vector design and will guide the derivation of complex adenovirus vectors from other serotypes.
Reverse-phase high-performance liquid chromatography (rp-HPLC)
Separation of proteins was accomplished on a 2 × 50 mm Jupiter 300Å C4 column (Phenomenex, Torrance, California, USA) at 0.2 ml per minute, elution by a gradient from 20 to 90% of acetonitrile in water, and trifluoroacetic acid at 0.1% was included throughout. Analytical and semi-preparative scale rp-HPLC was performed with an 1100 series Agilent LC system. Injection volumes varied between 5 μL with a maximum of 40 μL depending on viral particle concentrations. Ultraviolet absorbance at 210 nm was collected continuously throughout the HPLC analysis. The absorbance at 210 nm was normalized to absorbance at 360 nm to eliminate anomalous absorbance due to scatter. The fractions corresponding to peaks in the UV absorbance traces were frozen on dry ice for a minimum of a half-hour. The fractions were then lyophilized in a rotary lyophilizer. Using the procedures applied here minor structural proteins such as IVa2 or protease were either not observed or identifiable.
Whole protein mass spectroscopy by MALDI-TOF
The residue in the rp-HPLC fraction tubes that remained after lyophilization was re-suspended in 50% acetonitrile and 0.1% TFA typically in a volume of less than 10% of the original collected volume. A small volume of the sample was mixed with 20 mg/ml sinnapinic acid at a 1-to-1 ratio and 2 μL of this mixture was spotted and dried on a MALDI target plate along with protein molecular weight standards such as equine myoglobin and enolase. A dedicated target plate was used that had been optimized in the mass spectrometer to correct for slight deviations in the flatness of the plate. The machine was run in linear mode and delayed extraction of ca. 50-600 ns and acceleration voltages as required for maximum signal-to-noise and resolution was utilized. The fractioned proteins were also analyzed by SDS-PAGE allowing for co-identification of the individual proteins on both SDS-PAGE and HPLC.
Tryptic peptide mass spectroscopy fingerprints by MALDI-TOF
The dried rp-HPLC fractions were re-suspended in 50 μL of 25 mM ammonium carbonate and incubated with 0.1 μg of TPCK-treated trypsin at 37°C for 2 hours. The peptide mixture was mixed 1-to-1 with 10 mg/ml of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% TFA in water, spotted onto a MALDI target plate with peptide standards (Bradykinin and Angiotensin) and analyzed for a peptide fingerprint following calibration. Typically, the machine was run in reflector mode and utilized a delayed extraction of ca. 50-200 ns and acceleration voltages as required for maximum signal-to-noise and resolution. Monoisotopic masses from the MALDI-TOF machine were matched to proteins by Aldente (version 18/10/2004.) Searches were generally limited to include only those proteins isolated from viral sources but not to any specific virus family. The SDS-PAGE, rp-HPLC, and mass spectrophotometry analyses determined that purified stocks of wild type Ad35 contained intact and fragmented protein II (hexon).
Ad35 virus description
A lysate of Ad35 was obtained from the American Type Culture Collection (ATCC, catalog # VR-718D) and a viral genome cloned into a plasmid by homologous recombination . The wild type virus and recombinant derivatives were generated from the cloned genome on 293-ORF6 cells when liberated from the plasmid backbone. Sequencing of the entire viral genome revealed it to be nearly identical with the published Holden sequence found at GenBank accession number AY128640. All genome coordinates referenced in this manuscript are therefore based on the Holden sequence. Viral genomes were modified by homologous recombination in bacteria essentially as previously described [25, 26]. The CMV immediate early promoter and SV40 early polyadenylation signal expression cassette in the E1 region  is oriented to direct transcription toward the left ITR of the virus except for those viruses with the d2 deletion in which case it is in the opposite direction. The junctions of the Ad35 deletions are indicated in the text. The P5 promoter which directs pIX transcription in the Ad35E1(d5) configuration correspond to AAV bp 143-309 (ATCC accession number AF043303). The fiber shaft modified virus (Figure 1B) is an E1(d2) configuration with a bovine growth hormone polyadenylation signal and expressing HIV glycoprotein 140B in which Ad35 sequences 31,222-31,797 are replaced with Ad5 sequences 32,239-32,787 (GenBank accession number M73260.1) was compared to wild type virus.
Viral vector growth competitions
Virus with either E3 wild type (wt) or E3 deletions (X, HE) were competed pair wise against each other. All of the viruses contained a GFP expression cassette in the E1 (d8) deletion and are therefore E1-deleted or E1-, E3-deleted. Equal number of virus particles (pu) were mixed and 293-ORF6 cells were infected at 200 pu/cell total. 72 hours post infection cells were harvested and freeze thawed 3 times. Subsequently three sequential infections were carried out using 50 ul of crude lysate to infect a 60 cm plate of 293-ORF6 cells in a total volume of 500 ul before an additional 3.5 ml of DMEM with 10% FBS and 10 mM Zn Cl2 was added. The infections lasted 72 hrs. Relative levels of viral genomes were determined following the isolation of total DNA from the mixed input viral particles of a 200 μl aliquot of lysate using the High Pure Nucleic Acid Kit from Roche Applied Science (catalog # 11858874001). To distinguish the E3 wild type and E3 deletion X and wild type from deletion HE, the viral genomes were restricted with EcoRV and BlpI endonucleases (New England BioLabs), respectively, before being resolved on a 0.8% agarose gel using 0.5% TBE buffer.
Virion temperature lability
Wild type Ad35, an rAd35 with the E1 deletion d2 and GFP expression cassette, or an rAd35 with the E1 deletion d5 and HIV gp140BΔCFIΔV1V2 expression cassette were diluted in triplicate to 8 × 1010 particles/ml in 10 mM Tris pH7.5, 150 mM NaCl to a final volume of 800 ul in 1.5 ml microcentrifuge tubes on ice, then shifted to 48°C in a water bath. A 150 ul aliquot was removed at each time point, mixed with ice-cold 150 ul of DMEM + 5% FBS and kept on ice until tested for activity in the focus forming unit (FFU) assay. The data were presented as percentage of maximum activity because the input concentrations of the viruses were standardized to particle units and the three stocks of viruses had a 50-fold range in pu:FFU ratios.
Lambda cDNA library construction and cDNA sequencing
To construct the cDNA library total RNA was isolated using the PerfectPure RNA Cultured Cell Kit (catalog # 230240) from 5Prime using the human lung carcinoma cell line A549 24 hours post infection with Ad35 wild type at a moi of 5 FFU/cell. A lambda cDNA library was constructed by Lofstrand Labs Limited (USA) with a ZAP Express cDNA Synthesis Kit from Stratagene (catalog # 200403). Phage manipulation and screening were done essentially according to Current Protocols in Molecular Biology.
The sequence of the pIX and fiber cDNAs were determined. Phage containing pIX and fiber cDNA sequences were identified by probing plaque lifts using DNA labeled with the North2South Biotin Random Prime Labeling kit (catalog # 17075) from Thermo Scientific. The pIX and fiber DNA probes were generated by polymerase chain reaction (PCR) with primer pairs A35s3511 (GGAGTCTTCAGCCCTTATCTGACA) + A35a3853 (CGGCCACCTGCTGAGAAA) and A35s30987 (AACAACCACAGGCGGATCTCT) + A35a31619 (CTATCATAACTAGTCATGTAGTAACATATTCCATGAATGTAGTTTTC) respectively. The identified plaques were picked and phage expanded in culture. The phage lysates were used to prime PCRs whose products were gel purified and sequenced on an ABI 3100. The entire cloned cDNA was amplified using T3 (ATTAACCCTCACTAAAGGGA) and T7 (TAATACGACTCACTATAGG) flanking primers. In addition PCR products for the 5' and 3' portions of the cDNAs were also made and sequenced. The 5' specific reactions for pIX and fiber used A35a3853 and A35a31619 respectively along with the flanking primer CCGCGGATCCGCCCAGTACATGACCTTA. Similarly the cDNA 3' specific reactions used A35s3511 and A35s30987 in conjunction with the T7 primer for pIX and fiber, respectively.
Northern blot analysis
To generate viral RNA 293-ORF6 cells were infected at a moi of 5 FFU/cell when analyzing E1, pIX and fiber transcripts, and at a moi of 1 FFU/cell when analyzing E4 transcripts. Total RNA (5 ug) from viral and mock infected cells was harvested at 24 hours post infection except where noted in the manuscript, purified with the Versagene RNA cell Kit (Gentra Systems, catalog # VGR-0050CD), resolved on a 1 or 1.3% agarose gel containing 5% formaldehyde in 1 × MOPS buffer. The RNA was transferred to Immobilon-Ny+ (Millipore, catalog # INYC13750) rinsed sequentially in 6× SSC and water, air dried before being UV cross linked (UV Stratalinker 2400). The membrane was wetted with 5× SSPE and then prehybridized (50% Formamide, 2× SSC, 1% SDS, 5× Denhardt's and 0.5 mg/ml denatured salmon sperm DNA) at 42°C for 2-3 hours. Hybridization used the same buffer with 10% dextran sulphate overnight at 42°C along with the desired probe (see below) except for the strand specific probes which used the ULTRAhyb buffer (Ambion, catalog # AM8670). The membrane was washed sequentially twice briefly with 2× SSC, twice with 2× SSC, 0.1% SDS for 30 minutes at room temperature and three times with a solution of 0.5% SSC, 1% SDS at 56°C for 30 minutes each. The blot was air dried before being visualized using a Typhoon 9400 (Amersham Biosiences).
All probes were labeled with Redivue α-32P dCTP (Amersham, catalog # AA0075) using the Rediprime II Random Primer kit (Amersham, catalog # RPN1633) except for the strand specific probes which were DNA oligos labeled with γ-ATP using T4 kinase (New England BioLabs, catalog # M0201). The probes were: The description of the probes is as follows. The E1b and pIX probes 1-4 (Figure 3) were generated by PCR with primer pairs AGGAAGACTAGGCAACTGT + AAAGTAAGAAAAGCCACAG (probe 1), GGTGGTAATAGATACTCA + GCGTTGATGGGAAACAATATGCACAGTAGC (probe 2), CAAGCATGCCAGGTTCC + TGCGGGCAATAACCAAATGAT (probe 3), A35s3511 + A35a3853 (probe 4). The probe for the 2nd intron was generated from two oligos AGTATTGGGAAAACTTTGGGGTGGGATTTTCAGATGGACAGATTGAGTAAAAATTTGTTTTTTCTGTCTTG + CAAGACAGAAAAAACAAATTTTTACTCAATCTGTCCATCTGAAAATCCCACCCCAAAGTTTTCCCAATAC. The fiber probe was generated by PCR with primer pair A35s30987 + A35a31619. The E4 probes generated by PCR used primer pairs ATAGTAGCCTGACGAACAGGT + GCTGATGAGGCTTTGTATGTG (E4 Orf1), GACAAAATATCTTGCTCCTGT + GCAGACTAATTCAAAACTAAC (E4 Orf2), ACGTGGTAACTGGGCTCTGGT + TGCTTGAAACGCAATCCAGTT (E4 Orf4).
CACCTTCCCATTTGACAGAATA + TTGGGTTTGGCGGGATT (E4 Orf6). The E4 Orf3 probe is a subcloned DNA fragment comprising Ad35 base pairs 33258 through 33611. The E4Orf6 probes were an NruI to DraIII DNA restriction fragment corresponding to Ad35 coordinates 32,008 through 32,883. The strand specific probes are A35a3853, A35a31619 and ACAACGTGGTAACTGGGCTCTGGTGTAAGGGTGATGTCTGGCGCATGATG for pIX, fiber and E4, respectively.
Virus growth analysis
To determine the onset of viral replication, A549 cells were infected with wild type virus with 5 FFU/cell in triplicate using 60 cm plates. At the times indicated cells were washed 3× in PBS and total DNA isolated using the DNeasy Blood and Tissue kit from Qiagen (catalog # 69506). Viral genomes were quantified in a qPCR on an ABI 7700 using manufacture recommended conditions with primers CCCCGAATGGAAGTGCC and CGCTCTTTGGCGGCG along with probe 6FAMTGTTGAGTTGGACATACTCGCGTGCCMGBNFQ purchased from Applied Biosystems. The level of viral genome was normalized to ng of total DNA used in the reaction. The standard curve was generated with a plasmid of the complete E1-deleted Ad35 viral genome.
E4 complementation assays were carried out on PC-3 cells (Human prostate adenocarcinoma) infected with E1-wild type, E4-deleted viruses with 3 FFU/cell and harvested 72 hours post infection. Cells were freeze thawed 3 times and levels of active viral progeny were quantified by an FFU assay .
Generation of viral progeny was determined on 293-ORF6 cells which were infected with 1 FFU/cell. Cells were cultured in DMEM + 5%FBS + 100 um ZnCl2 and harvested 72 hours post infection. To quantify the number of particles cells were subjected to three freeze-thaw cycles, clarified through a 0.2 μm filter, and 100 ul volume was loaded onto a Poros 50D column (2 mm diameter, 10 cm length) equilibrated in 25 mM Tris pH 7.5. The column was washed with 25 mM Tris pH 7.5, vector eluted with 250 mM NaCl, 25 mM Tris pH 7.5, and particles detected by 214 nm absorbance. The number of viral particles was quantified by generating a standard curve using purified Ad35 particles standardized to A260 particle units . All of the viruses tested had either the GFP or enhanced GFP (eGFP) gene in the E1 expression cassette.
Vector packaging capacity
Limits on packaging capacity were assessed by testing for genetic rearrangements using a PCR. Viral genomes were liberated from its plasmid backbone by restriction digest, treated with a mixture of phenol:chlorophorm:isoamyl alcohol (24:24;1), ethanol precipitated and dissolved in water. A 60 mM plate of 293-ORF6 cells were transfected with 4 ug of viral genome using Poylyfect (Qiagen catalog # 301105). Five days post transfection cells were harvested, freeze thawed three times with 50 ul of lysate diluted to 500 ul with DMEM used to infect 60 cm plate of 293-ORF6 cells, rocked every 15 minutes for an hour before being fed with 4.5 ml DMEM + 5%FBS + 100 um ZnCl2. The virus was continued to be expanded using the same passaging regimen except cells were harvested at 72 hour post infection. To detect viral re-arrangement genomes were purified from 200 ul of lysates using the High Pure Viral Nucleic Acid kit (Roche, catalog # 11 858 874 001) in 20 ul of which 1 ul was used in a 25 ul PCR (94C 1 min, 30 cycles 94C 30 sec. × 55C 30 sec × 72C 3.5 min, with a final 72°C 10 min extension) with primers CGTACCGTGTCAAAGTCTTC and CGAGTTCTCAATGATCGAGA using Taq polymerase and Expand High Fidelity PCR buffer (Roch Diagnostics, catalog # 1435094 and 11732641001) and 1.7 ul resolved on an agarose gel.
Genetic stability analysis
293-ORF6 cells were transfected with the rAd35 genomic plasmids, cell - virus lysates were serial passaged 3 times (cytopathic effect appeared in the first passage), and viral DNA was extracted for a polymerase chain reaction (PCR) assay  using primers CGTACCGTGTCAAAGTCTTC and CGAGTTCTCAATGATCGAGA.
We are indebted to Nat Balsley, Angela Bommersbach, Andrew Glenn, Megan Gray, Richard Grier, Chi Hsu, Kurt Kroeger, David Rangel, Tatiana Simkova, and Idalia Yabe for excellent technical assistance.
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